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The occurrence of repetitive genomic changes that provide a selective growth advantage in pluripotent stem cells is of concern for their clinical application. However, the effect of different culture conditions on the underlying mutation rate is unknown. Here we show that the mutation rate in two human embryonic stem cell lines derived and banked for clinical application is low and not substantially affected by culture with Rho Kinase inhibitor, commonly used in their routine maintenance. However, the mutation rate is reduced by >50% in cells cultured under 5% oxygen, when we also found alterations in imprint methylation and reversible DNA hypomethylation. Mutations are evenly distributed across the chromosomes, except for a slight increase on the X-chromosome, and an elevation in intergenic regions suggesting that chromatin structure may affect mutation rate. Overall the results suggest that pluripotent stem cells are not subject to unusually high rates of genetic or epigenetic alterations. Mutations in human pluripotent stem cells (PSC) and whether any form during culture prior to use in a human clinical context are a concern. Here, the authors use hPSCs derived to cGMP standards and show they have low mutation rates after culture, noting this decreases on culturing in low (5%) oxygen conditions.

The application of human embryonic stem cell (hESC) derivatives to regenerative medicine is now becoming a reality. Although the vast majority of hESC lines have been derived for research purposes only, about 50 lines have been established under Good Manufacturing Practice (GMP) conditions. Cell types differentiated from these designated lines may be used as a cell therapy to treat macular degeneration, Parkinson’s, Huntington’s, diabetes, osteoarthritis and other degenerative conditions. It is essential to know the genetic stability of the hESC lines before progressing to clinical trials. We evaluated the molecular karyotype of 25 clinical-grade hESC lines by whole-genome single nucleotide polymorphism (SNP) array analysis. A total of 15 unique copy number variations (CNVs) greater than 100 kb were detected, most of which were found to be naturally occurring in the human population and none were associated with culture adaptation. In addition, three copy-neutral loss of heterozygosity (CN-LOH) regions greater than 1 Mb were observed and all were relatively small and interstitial suggesting they did not arise in culture. The large number of available clinical-grade hESC lines with defined molecular karyotypes provides a substantial starting platform from which the development of pre-clinical and clinical trials in regenerative medicine can be realised.

Background Cell therapies based on human pluripotent stem cells (hPSCs) are in clinical trials with the aim of restoring vision in people with age-related macular degeneration. The final cell therapy product consists of retinal pigment epithelium (RPE) cells differentiated from hPSCs. However, hPSCs recurrently acquire genetic abnormalities that give them an advantage in culture with unknown effects to the clinically-relevant cell progeny. One of the most common genetic abnormalities in hPSCs is the sub-karyotype 20q11.21 copy number variant, known to carry oncogenes. Understanding the impact of this variant on RPE differentiation and its potential for malignant transformation is crucial for the development of safe and effective cell therapies. Methods We monitored the RPE differentiation efficiency of hPSCs with or without the 20q11.21 variant. We then phenotyped the purified RPE cells for functionality, purity and tumorigenicity potential. Results We observed that 20q11.21 clones exhibited an enhanced differentiation capacity, developing pigmented foci at a higher rate and yield compared to normal clones. Gene expression analysis confirmed the upregulation of key RPE markers in 20q11.21 clones. The enhanced differentiation capacity of 20q11.21 clones was found to be dependent on the activity of BCL-XL, located within the amplicon. Furthermore, we demonstrated that 20q11.21-containing RPE cells displayed a mature phenotype, maintained long-term stability, and exhibited no malignant transformation capacity in vitro. Conclusion We demonstrated that gain of 20q11.21 enhances the speed and yield of RPE differentiation without compromising the phenotype of the derivatives. Finally, we discovered that 20q11.21-localised BCL-XL is important for RPE differentiation with potential non-canonical roles in retinal biology.

Background A major challenge for the clinical use of human pluripotent stem cells is the development of safe, robust and controlled differentiation protocols. Adaptation of research protocols using reagents designated as research-only to those which are suitable for clinical use, often referred to as good manufacturing practice (GMP) reagents, is a crucial and laborious step in the translational pipeline. However, published protocols to assist this process remain very limited. Methods We adapted research-grade protocols for the derivation and differentiation of long-term neuroepithelial stem cell progenitors (lt-NES) to GMP-grade reagents and factors suitable for clinical applications. We screened the robustness of the protocol with six clinical-grade hESC lines deposited in the UK Stem Cell Bank. Results Here, we present a new GMP-compliant protocol to derive lt-NES, which are multipotent, bankable and karyotypically stable. This protocol resulted in robust and reproducible differentiation of several clinical-grade embryonic stem cells from which we derived lt-NES. Furthermore, GMP-derived lt-NES demonstrated a high neurogenic potential while retaining the ability to be redirected to several neuronal sub-types. Conclusions Overall, we report the feasibility of derivation and differentiation of clinical-grade embryonic stem cell lines into lt-NES under GMP-compliant conditions. Our protocols could be used as a flexible tool to speed up translation-to-clinic of pluripotent stem cells for a variety of neurological therapies or regenerative medicine studies.

Establishing how to effectively manufacture cell therapies is an industry-level problem. Decentralised manufacturing is of increasing importance, and its challenges are recognised by healthcare regulators with deviations and comparability issues receiving specific attention from them. This paper is the first to report the deviations and other risks encountered when implementing the expansion of human pluripotent stem cells (hPSCs) in an automated three international site–decentralised manufacturing setting. An experimental demonstrator project expanded a human embryonal carcinoma cell line (2102Ep) at three development sites in France, Germany and the UK using the CompacT SelecT (Sartorius Stedim, Royston, UK) automated cell culture platform. Anticipated variations between sites spanned material input, features of the process itself and production system details including different quality management systems and personnel. Where possible, these were pre-addressed by implementing strategies including standardisation, cell bank mycoplasma testing and specific engineering and process improvements. However, despite such measures, unexpected deviations occurred between sites including software incompatibility and machine/process errors together with uncharacteristic contaminations. Many only became apparent during process proving or during the process run. Further, parameters including growth rate and viability discrepancies could only be determined post-run, preventing ‘live’ corrective measures. The work confirms the critical nature of approaches usually taken in Good Manufacturing Practice (GMP) manufacturing settings and especially emphasises the requirement for monitoring steps to be included within the production system. Real-time process monitoring coupled with carefully structured quality systems is essential for multiple site working including clarity of decision-making roles. Additionally, an over-reliance upon post-process visual microscopic comparisons has major limitations; it is difficult for non-experts to detect deleterious culture changes and such detection is slow.

A process called permitting allows nonunion crew to work on a production, but only once all available union members have been dispatched. Grappling with the implications of positive tests is an integral part of the industry these days. [...]when it come s to producer sign-offs, he pushes to meet in person since Zoom technology isn't conducive to the process when details such as paint color appear different on each screen. Before shutting down in March, the production had already added sanitizing stations on set and producers were urging crew to distance as much as possible; during the shutdown, they were paid for seven weeks of at-home time.

[...]costume designer Susan Lyall's work on director Aaron Sorkin's \"The Trial of the Chicago 7\" involved real-life activists following protests at the 1968 Democratic National Convention. On Regina King's \"One Night in Miami,\" costume designer Francine Jamison-Tanchuck faced the challenge of designing costumes for four frequently photographed civil-rights leaders - Sam Cooke, Malcolm X, Muhammad Ali and Jim Brown - within the context of a fictional story. [...]of the decade they set out to portray, the costume designers all had to strike the right balance between historical accuracy and creative interpretation.

Human stem cells have the potential to transform medicine. However, hurdles remain to ensure that manufacturing processes produce safe and effective products. A thorough understanding of the biological processes occurring during manufacture is fundamental to assuring these qualities and thus, their acceptability to regulators and clinicians. Leaders in both human pluripotent and somatic stem cells, were brought together with experts in clinical translation, biomanufacturing and regulation, to discuss key issues in assuring appropriate manufacturing conditions for delivery of effective and safe products from these cell types. This report summarizes the key issues discussed and records consensus reached by delegates and emphasizes the need for accurate language and nomenclature in the scientific discourse around stem cells. Novel therapies derived from different kinds of precursor cells and stem cells are increasingly moving to clinical trials to restore tissue function in patients who have suffered injury or disease. The manufacture of these new therapies is unusually complex, which means that the manufacturing processes require great attention to assure they are safe and effective. This paper describes a conversation among experts in the field who are exploring therapeutic applications of two different kinds of stem cells (pluripotent stem cells and tissue-derived stem/precursor cells). It considers critical issues in developing the manufacturing process for each of these quite different cells types.

The project brought to light previously unrecognized contributions of Native Americans in music, unidentified in part because of how the musicians once cloaked their heritage. In 2021, Halbritter was instrumental in presenting an Academy Museum event honoring the contributions of Native artists, including Robbie Robertson, Tantoo Cardinal and Wes Studi. Continuing to harness the power of Hollywood, Halbritter is developing a film about the Carlisle Indian School, an assimilation-minded boarding school in Carlisle, Pa., that future Olympian Jim Thorpe attended and has come under fire for harsh conditions.